Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2

The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1fnob2 mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1fnob2 mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts

Western blot of spleen samples from <i>Cacna1f^wt</i>, <i>Cacna1f^nob2</i>, and <i>Cacna1f^G305X</i> mice probed with a Ca_v1.4-specific antibody directed against the C-terminus of the channel.

<p>Full-length protein is visible in all lanes except for <i>Cacna1f^G305X</i>.</p

RT-PCR analysis of <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice.

<p>A. Schematic representation of the location of PCR primers used. Primers RR44, 45, and 46 were used for RT-PCR reactions; primers RR50, 51, 52, and 53 were used for genomic PCR reactions. B. Agarose gel depicting RT-PCR reaction products for mRNA isolated from <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice. Regardless of the primer pair used, only a single band is detected using mRNA from <i>Cacna1f^wt</i> mice. Using mRNA from <i>Cacna1f^nob2</i> mice, however, two bands are visible (see arrows). The relative intensities of the fluorescence signals indicate that the larger-M_r band accounts for ∼90%, and the smaller-M_r band for ∼10%, of the total mRNA.</p

Optokinetic Response Data for <i>Cacna1f^nob2</i> and <i>Cacna1f^wt</i> mice.

<p>Optokinetic Response Data for <i>Cacna1f^nob2</i> and <i>Cacna1f^wt</i> mice.</p

Sequencing results of <i>Cacna1f^wt</i> and <i>Cacna1f^nob2</i> mice.

<p>A. Sequencing results from the larger-M_r, more intense band. Sequences are given for the DNA (small letters) and corresponding protein (capital letters); the sequence corresponding to wild type Ca_v1.4 protein is highlighted in grey. The ETn transposable element is inserted after the ninth Ca_v1.4 amino acid, and encodes an in-frame stop codon (highlighted in red) which is predicted to result in truncation of the Ca_v1.4 protein after only 25 amino acids. The underlined ETn sequence highlights a repetitive sequence (compare to the beginning of the ETn sequence, beginning at amino acid number 4). B. Same as A., but for the smaller-M_r, less intense band. Note that the inserted ETn element is shorter, and the in-frame stop codon is missing. C. Alignment of the predicted N-terminal amino acid sequences of wild type Ca_v1.4 protein and Ca_v1.4 protein encoded in the smaller-M_r band. A region of approximately 22 amino acids differs in the N-termini of the two clones.</p

Primer sequences.

<p>Primer sequences.</p

Biophysical properties of Ca_v1.4^wt and Ca_v1.4^nob2 channels, coexpressed with β_2a and α₂–δ₁ subunits in tSA-201 cells.

<p>A. Representative current waveforms for Ca_v1.4^wt (left) and Ca_v1.4^nob2 (right) recorded with 20 mM Ba²⁺ external saline. Horizontal scale bars denote 25 ms, and vertical scale bars 25 pA. B. Average activation (filled symbols) and inactivation (hollow) symbols for Ca_v1.4^wt (squares) and Ca_v1.4^nob2 (circles) recorded with 20 mM Ba²⁺ external saline. Average activation parameters from 11 Ca_v1.4^wt cells and 13 Ca_v1.4^nob2 cells are: V_{act, wt} = −3±4 mV, V_{act, nob2} = −1±4 mV (n = 13); G_{max, wt} = 4±3 nS and G_{max, nob2} = 3±1 nS; S_wt = 9±1 mV and S_nob2 = 8.2±0.8 mV. These values are stastically identical, and are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone-0002538-t003" target="_blank">Table 3</a>. Average inactivation parameters from these cells are V_{inact, wt} = −18±11 mV and V_{inact, nob2} = −22±10 mV, with a large fraction of non-inactivating current for both channels. These values statistically identical and are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone-0002538-t003" target="_blank">Table 3</a>. C. Average half-inactivation potentials for channels recorded with 2 mM Ca²⁺ as charge carrier. Currents were substantially smaller than with 20 mM Ba²⁺, but were distinguishable from background noise, and were obtained using a ramp protocol identical to that previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone.0002538-Doering2" target="_blank">[31]</a>, obtained by ramping voltage from −100 mV to +100 mV over 500 ms. Values were V_{act, wt} = −17±8 mV (average peak current size −9±4 pA) and V_{act, nob2} = −17±6 mV (average peak current size −9±3 pA). The shift observed with switching from 20 mM Ba²⁺ to 2 mM Ca²⁺ as external charge carrier is similar to that we have previously reported for the human Ca_v1.4 channels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002538#pone.0002538-McRory1" target="_blank">[16]</a>.</p

A. Representative Coomassie Brilliant Blue-stained 10% PAGE gel from three separate experiments.

<p>N-termini from Ca_v1.4^wt or Ca_v1.4^nob2 were fused to glutathione S-transferase and purified on glutathione beads. Lysates from eye (lanes D–F) or spleen (G–I) were then incubated with the beads, and subsequently washed. Unpurified eye or spleen lysates (5 µL) or purified beads+lysates (40 µL) were loaded onto the gel. Lanes are as follows: A: unpurified eye, B: unpurified spleen, C: protein ladder, D: glutathione-sepharose beads+eye lysates, E: glutathione-sepharose beads+eye lysates+GST-Ca_v1.4 N-terminus, F: glutathione-sepharose beads+eye lysates+GST-Ca_v1.4^nob2 N-terminus, G: glutathione-sepharose beads+spleen lysates, H: glutathione-sepharose beads+spleen lysates+GST-Ca_v1.4 N-terminus, I: glutathione-sepharose beads+spleen lysates+GST-Ca_v1.4^nob2 N-terminus. A prominent band of M_r slightly larger than 37 kDa is observed in lanes E and H, but is absent from lanes F and I. This band was interpreted as a filamin A protein fragment. B. GST-fused N-termini from Ca_v1.4^wt or Ca_v1.4^nob2 were incubated with HA-tagged C-termini of filamin A or filamin B. Only GST-Cav1.4^wt N-terminus was capable of interacting with either filamin A or filamin B. Lanes 1-3 correspond to filamin A (with GST-Cav1.4^wt, GST-Cav1.4^nob2, and GST, respectively); lanes 4–6 correspond to filamin B (with GST-Cav1.4^wt, GST-Cav1.4^nob2, and GST, respectively); lanes 7 and 8 to filamin A and B, respectively (no GST construct); lanes 9–11 GST-Cav1.4^wt, GST-Cav1.4^nob2, and GST, respectively (with no filamin constructs); lane 12 is unpurified GST-Cav1.4^wt lysate; lane 13 is unpurified GST-Cav1.4^nob2 lysate.</p